超声介导叶酸靶向携氧载紫杉醇脂质微泡抑制裸鼠卵巢癌腹腔移植瘤的实验研究

发布时间：2018-01-27 13:02

  本文关键词： 微泡 叶酸 超声 巨噬细胞 卵巢癌　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一部分:合成叶酸靶向携氧载紫杉醇脂质微泡并检测体内寻靶能力第一节制备叶酸靶向携氧载紫杉醇脂质微泡及体内靶向能力分析目的:合成叶酸靶向携氧载紫杉醇脂质微泡,探究TOPLMBs的体内靶向能力。方法:建立裸鼠人卵巢癌SKOV3腹腔移植瘤模型,观察动物肿瘤模型的生长规律,获取肿瘤小体,HE染色,免疫组化法检测肿瘤组织中叶酸受体(FR)的表达情况。采用机械振动法分别制备携氧载紫杉醇脂质微泡(OPLMBs)及叶酸靶向携氧载紫杉醇脂质微泡(TOPLMBs),用Di I荧光标记。将成功建立卵巢癌模型的裸鼠随机分为OPLMBs组、TOPLMBs组和TOPLMBs+叶酸封闭组共3组。分别给予荧光标记的TOPLMBs、OPLMBs,在给药后30 min,24 h和48 h处死裸鼠,获得肿瘤腹水细胞及肿瘤小体;叶酸封闭组用叶酸阻断后加入荧光标记的TOPLMBs,给药后30 min同法获取腹水细胞及肿瘤小体,共聚焦显微镜观察荧光分布。取荷瘤裸鼠随机分为两组:TOPLMBs组和OPLMBs组,同法给予荧光标记的微泡,获取腹水细胞,流式细胞术分析腹腔卵巢癌细胞及巨噬细胞的荧光摄取情况。结果:裸鼠卵巢癌腹腔移植瘤模型成瘤率为100%,荷瘤裸鼠平均成瘤时间为8.2±0.84天,肿瘤组织高度表达叶酸受体。荧光显微镜观察切片:在给药后30min、24h及48h,TOPLMBs组的荧光分布明显多于OPLMBs组,TOPLMBs组在30min荧光表达最强。在给药后30min,叶酸封闭组荧光表达量微弱表达,但与OPLMBs组无明显差别。流式细胞术:巨噬细胞摄取荧光量TOPLMBs组5倍强于OPLMBs组,(P0.05),人卵巢癌SKOV3细胞摄取荧光量TOPLMs组3倍强于OPLMBs组(P0.05)。结论:人卵巢癌细胞株SKOV3腹腔移植瘤模型可成功建立,TOPLMBs与腹腔肿瘤小体及腹水中肿瘤细胞、肿瘤相关巨噬细胞能很好的结合,TOPLMBs能进入肿瘤组织内部。TOPLMBs有良好的体内寻靶能力。第二节:叶酸靶向携氧载紫杉醇脂质微泡在荷瘤裸鼠的组织分布目的:探讨叶酸靶向携氧载紫杉醇脂质微泡的组织分布情况,明确给药后超声辐照时间。方法:将荷瘤裸鼠随机分为3组:(a)紫杉醇组(PTX),(b)携氧载紫杉醇脂质微泡组(OPLMBs)(c)叶酸靶向携氧载紫杉醇脂质微泡组(TOPLMBs)。按紫杉醇20 mg/kg的药量腹腔给药。注射后30min处死裸鼠,迅速取出裸鼠腹腔肿瘤小体、腹腔淋巴结、腹水、血液,高效液相色谱法检测各组织中的药物浓度。结果:TOPLMBs组、OPLMBs组及PTX组肿瘤小体紫杉醇药量分别为(51.63±6.29μg/m L)、(20.56±5.39μg/m L)和(3.59±0.81μg/m L),腹腔淋巴结紫杉醇药量分别为(3.27±0.76μg/m L)、(1.20±0.12μg/m L)和(0.75±0.09μg/m L),血液中紫杉醇药量分别为(0.56±0.10μg/m L)、(1.19±0.12μg/m L)和(3.11±0.10μg/m L)。腹水中紫杉醇含量分别为(3.25±0.18μg/m L)、(6.51±0.13μg/m L)和(8.96±0.23μg/m L)。肿瘤小体、腹腔淋巴结中TOPLMBs组紫杉醇药量均显著高于OPLMBs组及PTX组(P0.05)。腹水、血液:TOPLMBs组紫杉醇浓度显著低于OPLMBs组及PTX组(P0.05)。结论:TOPLMBs能更好的将紫杉醇输送到肿瘤小体、腹腔淋巴结,在腹水、血液中的药物浓度小,TOPLMBs有可能提高抗肿瘤效果,减轻全身副作用。第二部分超声介导叶酸靶向携氧载紫杉醇脂质微泡治疗裸鼠卵巢癌腹腔移植瘤的研究目的:探讨超声介导叶酸靶向携氧载紫杉醇脂质微泡对裸鼠卵巢癌腹腔移植瘤生长抑制效应及相关机制。方法:将56只模型裸鼠随机分为7组:(a)对照组,(b)紫杉醇组,(c)紫杉醇+超声组,(d)携氧载紫杉醇脂质微泡组,(e)携氧载紫杉醇脂质微泡+超声组,(f)叶酸靶向携氧载紫杉醇脂质微泡组,(g)叶酸靶向携氧载紫杉醇脂质微泡+超声组,每组8只。最后一次治疗后24 h,随机处死3只获取肿瘤小体及腹水细胞,TUNEL检测肿瘤细胞凋亡,CD68免疫组化染色标记肿瘤相关巨噬细胞;免疫组化检测血管内皮生长因子(VEGF)及肿瘤微血管密度(MVD);流式细胞仪检测腹水中FR阳性细胞的凋亡;余下裸鼠观察荷瘤裸鼠的生存期。结果:从(a)组到(g)组的中位生存时间分别为:31天、37天、36天、31天、41天、32天和52天。与对照组(a)PBS组比较,PTX组、PTX+US组、OPLMBs+US组、TOPLMBs+US组均能显著延长荷瘤裸鼠生存期(P0.05),其中以TOPLMBs+US组延长生存时间最多(P0.05)。肿瘤组织中肿瘤细胞与肿瘤相关巨噬细胞凋亡:与PBS组比较,PTX组、PTX+US组、OPLMBs+US组、TOPLMBs+US组细胞凋亡率显著增高(P0.05),其中TOPLMBs+US组肿瘤细胞与肿瘤相关巨噬细胞凋亡率最高(P0.05)。肿瘤组织中VEGF表达及MVD生成情况:与PBS组相比,PTX组、PTX+US组、OPLMBs+US组、TOPLMBs+US组的VEGF及微血管密度明显降低(P0.05),以TOPLMBs+US组最低(P0.05)。从(a)组到(g)组的腹水中叶酸受体阳性细胞凋亡率分别为:(5.84±0.28)%,(18.72±3.44)%,(18.46±0.80)%,(7.63±0.90)%,(37.88±8.04)%,(7.34±1.08)%和(86.62±2.38)%,TOPLMBs+US组叶酸受体阳性细胞凋亡率显著高于各组(P0.05)。结论:超声介导叶酸靶向携氧载紫杉醇脂质微泡能显著延长荷瘤裸鼠的生存期,主要通过双靶向杀伤肿瘤细胞与肿瘤相关巨噬细胞、改善肿瘤乏氧微环境并抑制肿瘤血管生成来实现。
[Abstract]:The first part: the synthesis of folate targeted oxygen carrying paclitaxel carrying liposome microbubbles in vivo detection and targeting ability of the first control preparation of folic acid targeted paclitaxel load to the oxygen carrying alcohol lipid microbubbles and in vivo targeting ability analysis objective: to synthesize folate targeted oxygen carrying paclitaxel carrying liposome microbubbles, explore the body of TOPLMBs targeting ability methods: nude mice of human ovarian carcinoma SKOV3 transplanted tumor model, observe the growth of animal tumor model, obtaining tumor corpuscle, HE staining, folate receptor was detected by immunohistochemistry in tumor tissues (FR). The expression of the mechanical vibration method were prepared by oxygen carrying paclitaxel carrying liposome microbubbles (OPLMBs) and folate targeted oxygen carrying paclitaxel carrying liposome microbubbles (TOPLMBs), Di was labeled with I fluorescence. The ovarian cancer model of nude mice were randomly divided into OPLMBs group, TOPLMBs group and TOPLMBs+ folic acid group closed a total of 3 groups. The fluorescence labeled TOPLMBs were treated with OPLMBs. After administration, 30 min, 24 h and 48 h were sacrificed and the tumor ascites cells and tumor bodies; folic acid group closed after adding fluorescent labeled folic acid blocking TOPLMBs, 30 min after administration with ascites and tumor cells were obtained by confocal microscopy, fluorescence distribution from tumor bearing mice were randomly divided. Into two groups: TOPLMBs group and OPLMBs group, microbubble was given for the same fluorescent marker, obtain ascites cells, fluorescence uptake analysis of ovarian cancer cells and peritoneal macrophages by flow cytometry. Results: ovarian cancer xenograft in nude mice tumor was 100%, the average time of tumor formation in nude mice was 8.2. 0.84 days, tumor tissue high expression of folate receptor. Fluorescence microscopy sections: 30min after administration, 24h and 48h, the fluorescence distribution of TOPLMBs group was significantly higher than that of group OPLMBs, group TOPLMBs has the strongest expression in 30min fluorescence. 30min after administration of folic acid fluorescence blocking group The expression of weak expression, but no significant difference with OPLMBs group. Flow cytometry: macrophage uptake fluorescence group TOPLMBs, 5 times stronger than that of OPLMBs group (P0.05), human ovarian cancer SKOV3 cell uptake of fluorescence in TOPLMs group is 3 times stronger than that of OPLMBs group (P0.05). Conclusion: human ovarian cancer cell line SKOV3 in abdominal cavity transplantation tumor model can be successfully established, TOPLMBs and abdominal tumor bodies and ascites cells, tumor associated macrophages can be a very good combination, TOPLMBs can enter the tumor tissue in vivo.TOPLMBs has good targeting ability. Section second: folate targeted oxygen carrying paclitaxel lipid microbubbles in nude mice tissue Objective: to investigate the distribution of folate targeted oxygen carrying paclitaxel carrying liposome microbubbles, definite irradiation time after treatment. Methods: the tumor bearing mice were randomly divided into 3 groups: (a) paclitaxel group (PTX), (b) oxygen carrying paclitaxel carrying liposome microbubbles group (OPLMBs) (c) 鍙堕吀闈跺悜鎼烘哀杞界传鏉夐唶鑴傝川寰�娉＄唬�

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